A major bottleneck in virtually all areas of biomedical sciences and disease diagnoses is a paucity of high-quality affinity reagents. Affinity reagents are indispensable for delineating the molecular mechanisms of diseases, for detecting and characterizing cellular abnormalities, and for characterizing effects of drugs. In this post-genome era, the demand for high-quality affinity reagents is rapidly increasing across all fields of biomedical science.
Short peptide motifs are, in principle, attractive targets against which affinity reagents can be generated. Short peptides derived from a target protein or a synthetic peptide can be chemically synthesized, and the epitope or affinity ligand (the region that is recognized by an affinity reagent) can be readily deduced. Short peptide motifs (and their modification state) are not only indicators (or biomarkers) of the functional state of extremely important components of these networks, but can be used in various other assays and methodologies as affinity ligands.
Currently, antibodies are the gold standard of affinity reagents. However, making antibodies that recognize a particular short peptide motif with high affinity and specificity is difficult and time-consuming. The paucity of good antibodies to short peptides is not due to a lack of intensive effort. The difficulty arises from the fundamental thermodynamics of the binding of a short flexible peptide motif in which a small number of antibody-motif contacts must compensate for a large loss of conformational entropy. This is, perhaps, not so surprising because antibodies have not evolved specifically to bind short peptide motifs.
Antibodies have additional serious limitations. Monoclonal antibody production is low throughput and expensive and polyclonal antibodies have a fundamental problem in production scalability and archiving. Although many monoclonal antibodies to whole protein antigens exist, very few are available for defined short peptide motifs.
As to polyclonal antibodies, which are widely used, the upfront costs and efforts to generate such antibodies are low. Polyclonal antibodies do not, however, meet the criteria for high-performance affinity reagents. A polyclonal antibody is impossible to reproduce with the identical properties once the stock is depleted, making it unfeasible to establish a robust standard assay that can be broadly distributed. Further, the inherently heterogeneous nature of a polyclonal antibody makes it impossible to define precisely its properties such as motif specificity and affinity. Polyclonal antibodies also cannot be easily reformatted for different applications.
In the last decade, nucleic acid-based affinity reagents have been developed (Rimmele, 2003; Yan et al., 2005). However, they share the same difficulty as antibodies in generating high-affinity binders to short peptide motifs. To date, no nucleic acid aptamers have been generated that have low nM Kd to a short peptide motif derived from a natural protein.
Each of the current approaches for designing affinity reagents has numerous disadvantages and fails to generate high-performance affinity reagents to small peptide motifs that are critically important. There remains a need for additional affinity reagents.